Physiol. Genomics Genetics/Genomics of Vascular Disease Workshop
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 31: 281-294, 2007. First published July 31, 2007; doi:10.1152/physiolgenomics.00098.2007
1094-8341/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
31/2/281    most recent
00098.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, H. S.
Right arrow Articles by Jetten, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, H. S.
Right arrow Articles by Jetten, A. M.
Received 30 April 2007; accepted in final form 30 July 2007.
Physiological Genomics 31:281-294 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Gene expression profiling reveals a regulatory role for ROR{alpha} and ROR{gamma} in phase I and phase II metabolism

Hong Soon Kang1,*, Martin Angers1,*, Ju Youn Beak1, Xiying Wu2, Jeffrey M. Gimble2, Taira Wada3, Wen Xie3, Jennifer B. Collins4, Sherry F. Grissom4 and Anton M. Jetten1

1 Cell Biology Section, National Institutes of Health, Research Triangle Park, North Carolina
4 Microarray Group, Division of Intramural Research, The National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
2 Stem Cell Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
3 Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Retinoid-related orphan receptors alpha (ROR{alpha}) and gamma (ROR{gamma}) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The ROR{alpha}1 and ROR{gamma}1 isoforms, but not ROR{alpha}4, show an oscillatory pattern of expression during circadian rhythm. To obtain insight into the physiological functions of ROR receptors in liver, we analyzed the gene expression profiles of livers from WT, ROR{alpha}-deficient staggerer (sg) mice (ROR{alpha}sg/sg), ROR{gamma}–/–, and ROR{alpha}sg/sgROR{gamma}–/– double knockout (DKO) mice by microarray analysis. DKO mice were generated to study functional redundancy between ROR{alpha} and ROR{gamma}. These analyses demonstrated that ROR{alpha} and ROR{gamma} affect the expression of a number of genes. ROR{alpha} and ROR{gamma} are particularly important in the regulation of genes encoding several phase I and phase II metabolic enzymes, including several 3ß-hydroxysteroid dehydrogenases, cytochrome P450 enzymes, and sulfotransferases. In addition, our results indicate that ROR{alpha} and ROR{gamma} each affect the expression of a specific set of genes but also exhibit functional redundancy. Our study shows that ROR{alpha} and ROR{gamma} receptors influence the regulation of several metabolic pathways, including those involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that RORs are important in the control of metabolic homeostasis.

liver; nuclear receptor; metabolism; sulfotransferase; staggerer mice; gene expression analysis; circadian rhythm; retinoid-related orphan receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE RETINOID-RELATED ORPHAN RECEPTOR (ROR) subfamily of nuclear receptors consists of three members: alpha (ROR{alpha}), beta (RORß), and gamma (ROR{gamma}), also referred to as NR1F1–3 or RORA–C, respectively (16, 17). Through alternative splicing and promoter usage, each ROR gene generates several variants that are expressed in a tissue-specific manner and regulate specific target genes and physiological functions. These variants have been implicated in the regulation of distinct biological processes. ROR receptors preferably bind as monomers to specific ROR response elements (ROREs) (11, 29) in the regulatory region of target genes. Repression and activation of gene transcription by RORs are mediated through the recruitment of co-repressors and co-activator complexes, respectively, as has been demonstrated for other nuclear receptors (16, 17). Recent crystal structure analyses of RORs have provided evidence indicating that the transcriptional activity of RORs is ligand dependent (20, 39). Cholesterol-sulfate and other (sulfated) lipid metabolites have been reported to function as agonists for ROR{alpha}, while several retinoids were demonstrated to bind RORß and ROR{gamma} and to act as partial antagonists (20, 39).

Characterizations of mice deficient in the expression of RORs have implicated ROR{alpha}, RORß, and ROR{gamma} in the control of distinct physiological processes. These studies showed that ROR{alpha} plays a critical role in the maturation and survival of Purkinje cells (4, 9, 12, 17). ROR{alpha} has also been implicated in bone formation, lipid homeostasis, and in the regulation of several immune functions (7, 15, 30, 32, 37, 43). ROR{alpha}, ß, and {gamma} all appear to have a role in the regulation of circadian rhythm (2, 17, 34). Moreover, a recent study implicated RORß in the regulation of the S opsin gene in retinal cone photoreceptors (36). ROR{gamma}t (also referred to as ROR{gamma}2) has been reported to play a critical role in the development of secondary lymphoid tissues and in thymopoiesis (10, 17, 25, 41, 42).

While the expression of RORß is rather restricted and shows little overlap with those of the other two ROR receptors, ROR{alpha} and ROR{gamma} are coexpressed in many tissues, including brown fat, liver, and kidney (17). The latter raised the question whether there is any functional redundancy between these two receptors as has been demonstrated for several other nuclear receptors. To obtain greater insight into this question and the roles of RORs in liver, we generated ROR{alpha} and ROR{gamma} double knockout (DKO) mice and compared the gene expression profiles of livers from wild-type (WT), ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice by microarray analysis. These analyses showed that lack of ROR{alpha} and ROR{gamma} expression affects the expression of several genes encoding phase I and phase II metabolic enzymes. In addition, our data demonstrated that ROR{alpha} and ROR{gamma} each can influence the expression of a specific set of genes but also exhibit functional redundancy and that they can affect gene expression in a positive as well as negative manner. Our study shows that ROR{alpha} and ROR{gamma} receptors affect the expression of several genes involved in the metabolism of steroids, bile acids, and xenobiotics, suggesting that they may play an important role in the control of metabolic homeostasis and the detoxification and elimination of endogenous and exogenous compounds.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals.
Heterozygous C57BL/6 staggerer (ROR{alpha}+/sg) mice were purchased from Jackson Laboratories (Bar Harbor, ME). The staggerer (ROR{alpha}sg/sg) mice, a natural mutant mouse strain, contain a 6.5-kb deletion in the ROR{alpha} gene resulting in a functional knockout of ROR{alpha} (13). C57BL/6 ROR{gamma}–/– mice were described previously (25). ROR{alpha}sg/sgROR{gamma}–/– (DKO) mice were generated by crossing ROR{alpha}+/sg with ROR{gamma}–/– mice. ROR{alpha} genotyping was carried by polymerase chain reaction (PCR) of tail DNA according to the instructions of Jackson Laboratories while genotyping of the ROR{gamma}–/– mice was performed as previously described (25). Littermate WT mice were used as controls. The animals were bred at NIEHS and were supplied ad libitum with NIH-A31 formula and water. Blood was collected by heart puncture, and sera were stored at –80°C. The C57BL/6 and AKR/J mice used in the study were maintained on a constant 12-h light/12-h dark cycle with the light cycle beginning at 6 AM. All animal protocols followed the guidelines outlined by the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the National Institute of Environmental Health Sciences (NIEHS) and the Pennington Biomedical Research Center.

RNA isolation.
Tissues were collected from mice at the circadian time (CT) indicated. Tissues were rapidly dissected, and parts were either processed in RNA Later solution (Ambion, Austin, TX) or flash frozen in liquid nitrogen. Tissues were stored at –80°C. Tissues were then homogenized in 4 ml of RLT solution in presence of ß-mercaptoethanol with a Polytron PT 3000 (Brinkman Instruments, Westbury, NY). The homogenate was loaded into a QIAshredder column (Qiagen, Valencia, CA) and centrifuged at 12,000 g for 3 min. The supernatant was collected, and one volume of 70% ethanol (50% for liver) was added. The mixture was then loaded onto an RNeasy midi-column and RNA isolated following the manufacturer's instructions (Qiagen). The quality and integrity of the RNA were assessed by bioanalyzer (Agilent, Santa Clara, CA) and agarose gel electrophoresis.

Northern blot analysis.
Northern blot analyses were performed as described (21). Briefly, 15 µg of total RNA were separated on a 1.2% agarose gel containing 0.5% formaldehyde in 1x MOPS buffer, then transferred onto a nylon membrane (Sigma, St. Louis, MO). After UV-cross-linking, the membrane was hybridized to [32P]-labeled probes for Sult1e1, Sult2a1, insulin growth factor binding protein 1 (Igfbp1), Elovl3, Keg-1, Hsd3b5, and Cyp2b10. The membrane was then washed and exposed on Hyperfilm (Amersham Bioscience) at –70°C.

Microarray analysis.
Gene expression analyses were conducted by the NIEHS Microarray Group on Agilent mouse 20,000-oligo chips. Total RNA was isolated from livers of 8- to 12-wk-old WT, DKO, ROR{alpha}sg/sg, or ROR{gamma}–/– mice ~CT19. Each analysis was performed in duplicate, employing a fluor reversal. In the case of WT and DKO mice, two independent experimental replicates were analyzed with a different subset of mice. Briefly, equal amounts of total RNA from individual male mice (5 WT vs. 8 DKO in the first analysis, 4 WT vs. 4 DKO in the second analysis, 4 WT vs. 4 ROR{gamma}–/–, and 4 WT vs. 4 ROR{alpha}sg/sg) were pooled for each genotype group and then amplified using Agilent Low RNA Input Fluorescent Linear Amplification Kit. Starting with 0.5 µg of amplified total RNA, Cy3- or Cy5-labeled cRNA was produced according to manufacturer's protocol. For each two-color comparison, 750 ng of each Cy3- and Cy5-labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit. Hybridizations were performed for 16 h in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data were retrieved with the Agilent Feature Extraction software (v7.5), using defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver system (version 5.1) (Rosetta Biosoftware, Kirkland, WA). The Resolver system combines ratio profiles to create ratio experiments using an error-weighted average as described (45). P values are generated and propagated throughout the system and represent the probability that a given gene is significantly expressed. Genes with a P value <0.001 were considered statistically, differentially expressed. The signature genes were sorted into different categories with the help of the GeneSpring software. One microarray analysis was carried out with pooled RNA from livers of one female and two male WT mice vs. three female DKO mice. Except for several sex-specific genes, this analysis confirmed the pattern of differentially expressed genes obtained with RNA from livers of male WT and DKO mice (data not shown). The microarray data discussed in this study have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE7564.

Quantitative real-time PCR.
SYBRG and TaqMan quantitative real-time PCR (QRT-PCR) analyses were performed to validate several of the genes identified by microarray analyses and to measure gene expression as a function of CT. Total RNA from three individual mice within each genotype, sex, or CT group was analyzed as indicated. The RNA was reverse-transcribed using the high-capacity cDNA archive kit according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). QRT-PCR reactions with 50 ng of cDNA were carried out in triplicate in a 7300 Real Time PCR system (Applied Biosystems) as follows: 2 min at 50°C, 10 min at 95°C, then 40 cycles each at 95°C for 15 s, and 60°C for 60 s. Predesigned Assays-on-Demand primers/probe sets were purchased from Applied Biosystems: Mm00484132_m1 (Cyp4a14), Mm00484157_m1 (Cyp7b1), Mm00431814_m1 (ApoA4), Mm00468164_m1 (Elovl3), Mm00657677_mH (Hsd3b5). Mm00833447_m1 (Igfbp1), Mm00649796_m1 (Slco1a1), Mm00499178_m1 (Sult1e1). Others primers and probes (Table 1) were designed using the ABI PrimerExpress 2.0 software and synthesized by Sigma/Genosys. All results were normalized relatively to the 18S transcript except in the circadian rhythm experiments where cyclophilin B was utilized instead.


View this table:
[in this window]
[in a new window]

  		Table 1. List of primers and probes used for quantitative real-time-PCR

 
Sulfotransferase and steroid assays.
Flash-frozen liver tissues were homogenized with a Polytron at 4°C in 4 volumes of homogenizing buffer (250 mM sucrose, 5 mM HEPES, and 2 mM ß-mercaptoethanol, pH 7.4). The homogenates were centrifuged at 16,000 g for 10 min at 4°C. Subsequently the supernatants were collected and analyzed for sulfotransferase (Sult) activity following a modified protocol adapted from Ref. 22. Briefly, reaction buffer [100 mM Tris, 10 mM MgCl2, 20 mM of substrate, and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), pH 7.4] was added to protein extracts (100 µg) and incubated for 30 min at 37°C. [2,4,6,7-3H]-labeled estradiol (PerkinElmer Life Sciences, Boston, MA) served as a substrate for Sult1e1 while [1,2,6,7-3H]-labeled dehydroepiandrosterone (DHEA, PerkinElmer Life Sciences) was used for Sult2a1. The enzymatic reactions were stopped by addition of 600 µl of Tris·HCl, pH 8.7, and 1.7 ml of dichloromethane. The samples were mixed thoroughly then centrifuged at 600 g for 5 min. The supernatant was collected, and the radioactivity was determined in a scintillation counter.

Estradiol and DHEA levels were determined by radioimmune assay (RIA). An estradiol RIA kit was purchased from Diagnostic Products (Los Angeles, CA), and a DHEA RIA kit was obtained from Diagnostic Systems Laboratory (Webster, TX). Assays were performed in an Apex Automatic gamma counter (ICN Micromedic Systems, Huntsville, AL).

Transfection.
Mouse primary hepatocytes were plated in six-well dishes and maintained in hepatocyte maintenance medium (Cambrex BioScience, Walkersville, MA) and then transfected with 4 µg of VP-ROR{alpha} or ROR{alpha} expression plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). VP-ROR{alpha} was created by fusing the VP16 activation domain of the herpes simplex virus to the amino terminus of human ROR{alpha}. Compared with its WT counterpart, VP-ROR{alpha} showed a similar affinity to prototypical RORE but exhibited substantially higher activity on RORE reporter gene (data not shown). Hydrodynamic liver transfection in 4-wk-old CD-1 female mice was carried out as described previously (48). Mice were killed, and liver tissues were harvested 6 h after the injection of VP-ROR{alpha} or ROR{alpha} plasmid DNA (5 µg). Total RNA was extracted and subjected to QRT-PCR analysis.

Blood analysis.
The levels of glucose, cholesterol, triglycerides, and HDL were determined using the Cobas Mira Classic Chemistry System (Roche Diagnostics Systems, Montclair, NJ), and the chemical reagents for all assays were purchased from Equal Diagnostics (Exton, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Generation and characterization of DKO mice.
Previous studies have shown that ROR{alpha} and ROR{gamma} are expressed in several tissues (5, 17). Some tissues, including liver, express both receptors, whereas other tissues, such as brain, express only ROR{alpha} (Fig. 1A). In addition to the generation of different isoforms, each ROR gene produces multiple transcripts of different sizes that are generated by the use of alternative polyadenylation signals (17). Mouse liver expresses the ROR{alpha}1 and ROR{alpha}4 isoforms and only the ROR{gamma}1 isoform (5, 16). Figure 1B shows that ROR{alpha}1 and ROR{gamma}1 exhibit an oscillatory pattern of expression consistent with a circadian rhythm (46). Both ROR{alpha}1 and ROR{gamma}1 mRNA are optimally expressed between CT16 and CT0 and expressed at low levels between CT4 and CT12. The expression of ROR{alpha}4 mRNA did not display a significant oscillatory expression pattern.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Expression of the retinoid-related orphan receptor (ROR){alpha} and ROR{gamma}. A: ROR{alpha} and ROR{gamma} are both highly expressed in liver. The expression levels of ROR{alpha} and ROR{gamma} mRNA in several mouse tissues were examined by Northern blot analysis. Expression of 18S rRNA served as loading control. *Transcripts of different size generated by the use of alternative polyadenylation signals. B: circadian oscillation of the expression of ROR{alpha}1, ROR{alpha}4, and ROR{gamma}1 mRNA in liver. The mRNA expression was examined by QRT-PCR analysis and normalized relative to cyclophilin B. Data from a single 24-h period were double plotted. Male AKR/J mice used in the study were maintained on a constant 12-h light/12-h dark cycle as indicated by the bar. CT, circadian time.

 
To obtain greater insight into the physiological functions of ROR{alpha} and ROR{gamma} receptors in liver and to determine whether ROR{alpha} and ROR{gamma} have redundant functions, we generated ROR{alpha}sg/sgROR{gamma}–/– DKO mice (Fig. 2A). The DKO mice were viable and exhibited all the main phenotypic characteristics previously found to be associated with ROR{alpha} and ROR{gamma} knockout mice including the staggerer phenotype, a characteristic of ROR{alpha}sg/sg mice, and the absence of lymph nodes and a greatly reduced thymus, characteristics of ROR{gamma}–/– mice (10, 12, 17, 25, 41). DKO mice weighed ~30% less than WT littermate controls, similar to the 25% reduction in body weight observed for ROR{alpha}sg/sg mice. No significant differences were observed in relative liver weights between each group (Fig. 2B), and histopathological examination did not reveal any obvious morphological changes in the liver of either ROR{alpha}sg/sg, ROR{gamma}–/–, or DKO mice (data not shown). A significant reduction in blood triglyceride (35%) and cholesterol (25%) levels was observed in DKO mice that was comparable to those observed in ROR{alpha}sg/sg mice, while triglyceride and cholesterol levels were not significantly changed in ROR{gamma}–/– mice (Fig. 3). The level of blood glucose was reduced by 25% in DKO mice compared with WT, and although a 12% reduction in blood glucose was observed in ROR{gamma}–/– mice, this reduction was not statistically different from that of WT mice. ROR{alpha}sg/sg and WT mice did not exhibit a significant difference in blood glucose levels (Fig. 3).


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 2. Characterization of ROR{alpha}sg/sg ROR{gamma}–/– double knockout (DKO) mice. A: genotyping of DKO, ROR{alpha}sg/sg, and ROR{gamma}–/– mice. Tail DNA was isolated and genotype examined by RT-PCR analysis. PCR products were separated by the gel-electrophoresis. The size of the PCR products of the wild-type (WT) and recombinant (Rec) allele for ROR{alpha} are 318 bp and 450 bp, respectively, while those of the WT and recombinant allele for ROR{gamma} are 380 bp and 430 bp, respectively. B: average total body weight. C: average relative liver and kidney weights of 3-mo-old male WT, ROR{alpha}sg/sg, and ROR{gamma}–/–, and DKO mice (for each group, n = 6).

 

Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Comparison of the levels of triglycerides, cholesterol, HDL, and glucose in WT, ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice; N = 24 (WT), 17 (DKO), 6 (ROR{alpha}sg/sg), and 13 (ROR{gamma}–/–). *P < 0.001; **P < 0.05; ***P > 0.05.

 
ROR{alpha} and ROR{gamma} have specific and redundant functions and act as positive and negative regulators of gene expression.
To obtain insight into the physiological functions of ROR receptors in liver, we compared the gene expression profiles from livers of WT, ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice by microarray analysis. Because ROR{alpha}1 and ROR{gamma}1 expression displayed a circadian rhythmicity, the tissues were collected at CT19, a time at which receptor expression is at its zenith. Differentially expressed genes were considered for further analysis if they were induced or repressed at least 1.5-fold or more in one of the three knockout groups and if their P value was <0.001. The gene profiling analysis indicated that in mouse liver, many genes were regulated by ROR{alpha} and/or ROR{gamma}. The expression of 69 genes was changed in both ROR{alpha}sg/sg and DKO mice, 12 genes in both ROR{gamma}–/– and DKO mice, 20 genes in ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice, while 797 genes were changed only in DKO mice. Table 2 shows a partial list of genes regulated by RORs in liver. The complete listing of all the changes in gene expression identified in the different comparisons is available at http://dir.niehs.nih.gov/microarray/jetten/home.htm. The genes most dramatically repressed or induced in liver from DKO mice, include genes encoding several Sults, cytochrome P450 (Cyp) enzymes, microsomal enzymes, hydroxysteroid dehydrogenases, glutathione transferases, and solute carriers. In addition, these data suggested that certain genes are regulated preferentially by either ROR{alpha} or ROR{gamma}, while other genes are under the control of both receptors, and indicate that ROR{alpha} and ROR{gamma} have specific as well as redundant functions in liver. The gene profiling analysis also showed that ~30% of the genes were induced, while ~70% were downregulated in livers from DKO-deficient mice. The latter indicates that RORs can function as positive as well as negative regulators of gene expression.


View this table:
[in this window]
[in a new window]

  		Table 2. Partial list of genes repressed or induced in livers of male DKO, ROR{alpha}sg/sg, and ROR{gamma}–/– mice

 
Expression profiling analyses reveal novel biological functions of ROR receptor.
RORs regulate the expression of genes encoding proteins with widely different functions including the cell adhesion protein cadherin 1, the mitochondrial enzyme creatine kinase (Ckmt2), Igfbp1, the lipophilic ligand carrier protein lipocalin 13 (Lcn13), the selenium binding protein 2 (Selenbp2), the asparagine synthetase (Asns), and the chemokine Ccl5, also named RANTES. Interestingly, among the genes up- or downregulated in liver from DKO mice compared with WT littermates were many genes encoding phase I and phase II proteins involved in the metabolism of lipids, steroids, and xenobiotics. The phase I genes regulated by RORs included several Cyp genes. The expression of Cyp2b9, Cyp2b10, Cyp39a1, Cyp4a14, and Cyp2b13 was induced, whereas that of Cyp7b1, Cyp2f2, and Cyp8b1 was repressed in liver of ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice. The expression of Cyp2b9, Cyp39a1, and Cyp7b1 appeared to be dependent on ROR{alpha} rather than ROR{gamma}. Cyp7b1, Cyp39a1, and Cyp8b1 have important functions in the biosynthesis of bile acids, and their regulation by RORs suggests a role for these receptors in the control of bile acid biosynthesis. The expression of two other P450 genes, Cyp7a1 and Cyp27a1, with major roles in the metabolism of cholesterol into bile acids, was not significantly altered between livers of WT and ROR-deficient mice (data not shown). Cyp2b9 and Cyp2b10 play a role in the oxidation of various xenobiotics and drugs, while Cyp4a10 and Cyp4a14 have been implicated in lipid hydroxylation (40). These observations suggest a role of RORs in the regulation of the metabolism of lipids, steroids, and xenobiotics.

RORs also affect the expression of several genes encoding phase II metabolic enzymes. These include several Sults, which catalyze the transfer of sulfonyl groups using PAPS as sulfate donor. The expression of Sult1e1 mRNA was greatly induced in liver of male DKO and ROR{alpha}sg/sg mice (Table 2). Loss of ROR{gamma} expression had little effect on Sult1e1 expression. These observations suggest that ROR{alpha} negatively regulates the expression of Sult1e1. Hydroxysteroid sulfotransferase Sult2a1 expression, which has been reported to be sex specific and only expressed in liver from female mice (33), was greatly induced in liver from DKO mice suggesting that is regulated by both ROR{alpha} and ROR{gamma}. Sult2a1 catalyzes the sulfonation of procarcinogens, xenobiotics, hydroxysteroids, and bile acids, particularly lithocholic acid (LCA), while Sult1e1 catalyzes the sulfonation of estrogen and estrone (23). These observations suggest a role for RORs in the regulation of the metabolism of steroids, bile acids, and xenobiotics.

Glutathione transferases form another group of phase II metabolic enzymes that catalyze the conjugation of glutathione with a wide variety of xenobiotics generally resulting in their detoxification and elimination (28). ROR deficiency inhibited the expression of several glutathione transferases, while the expression of others was enhanced (Table 2). These results further support a role of RORs in the modulation of xenobiotic metabolism.

Table 2 also shows that RORs regulate the expression of several members of the 3ß-hydroxysteroid dehydrogenase (Hsd3b) family. In mice the Hsd3b family consists of six members Hsd3b1–6. Hsd3b4 and Hsd3b5 are NADPH-dependent 3-ketosteroid reductases, while Hsd3b2, Hsd3b3, and Hsd3b6 are NAD+-dependent dehydrogenases/isomerases (35). The expression of Hsd3b2–6 was downregulated in DKO mice (Table 2). Hsd3b5, which is expressed largely in liver of male mice, was the most affected by the loss of ROR expression. The expression of Hsd3b5 and Hsd3b4 was downregulated in both ROR{alpha}- and ROR{gamma}-deficient mice and almost totally repressed in DKO mice. These results suggest that Hsd3b4 and Hsd3b5 are positively regulated by both ROR{alpha} and ROR{gamma}. Hsd3b family members catalyze either the biosynthesis of active steroid hormones or the inactivation of steroid hormones (35). Therefore, their regulation by RORs suggests a role for these receptors in the control of steroid hormone metabolism.

Northern blot and QRT-PCR analysis.
The induction or repression of the expression of Sult1e1, Sult2a1, Igfbp1, Elovl3, Keg-1, Hsd3b5, and Cyp2b9/10 mRNAs in livers from ROR-deficient mice was verified by Northern blot analysis (Fig. 4A). In addition, the expression of several genes was quantified by QRT-PCR (Fig. 4B). To obtain an independent evaluation, QRT-PCR analysis was performed on RNA samples that were different from the ones used in the microarray and Northern blot analysis. A good correlation was observed between QRT-PCR analysis and the data obtained by Northern blot and microarray analysis. The data confirmed that expression of Sult1e1 was induced in livers from both DKO and ROR{alpha}sg/sg mice but not greatly altered in ROR{gamma}–/– mice. This suggests that Sult1e1 is affected preferentially by ROR{alpha}. In contrast, the expression of Elovl3 was significantly decreased in DKO and ROR{gamma}–/– mice and somewhat decreased in ROR{alpha}sg/sg mice, suggesting that Elovl3 is modulated preferentially by ROR{gamma}. Expression of Cyp7b1 was preferentially regulated by ROR{alpha}. Hsd3b5 mRNA expression was repressed in both ROR{alpha}sg/sg and ROR{gamma}–/– mice, but its expression was considerably more repressed in livers from DKO mice, suggesting that both ROR{alpha} and ROR{gamma} affect Hsd3b5 expression and that loss of both RORs results in the maximal repression of this gene. Inversely, the expression of Sult2a1, Igfbp1, and Cyp4a14 is induced to a greater extent in DKO mice than in single knockout mice, suggesting that both receptors are involved in their negative regulation. The results with Hsd3b5, Cyp4a14, Sult2a1, and Igfbp1 expression are in agreement with the conclusion that ROR{alpha} and ROR{gamma} exhibit redundant functions. The expression of several genes, including Sult2a1 and Elovl3, has been shown to be sex dependent. RORs affect the expression of those genes only in females or males (33).


Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 4. Lack of ROR{alpha} and ROR{gamma} affects the expression of several genes. A: livers from 2 individual 8- to 12-wk-old male or female mice within each genotype group were collected at CT19. RNA was isolated and subsequently examined by Northern blot analysis using radiolabeled probes for Sul1e1, Sult2a1, Cyp2b9/10, Hsd3b5, Igfbp1, Elovl3, Keg-1. B: comparison of gene expression in livers from WT, DKO, ROR{alpha}sg/sg, and ROR{gamma}–/– mice by quantitative real-time (QRT)-PCR analysis. Livers of 3 individual 8- to 12-wk-old male or female mice within each genotype group were collected at CT19. RNA was isolated and the expression of a selected group of differentially expressed genes identified by gene profiling analysis analyzed by QRT-PCR. *P < 0.05 (compared with WT).

 
Increased Sult1e1 and Sult2a1 expression and activity in DKO mice.
The induction of Sult1e1 and Sult2a1 expression in ROR-deficient mice was confirmed by QRT-PCR analysis (Fig. 5A). Sult2a1 activity was only detectable in livers from female mice as reported previously (33). The results further demonstrated that Sult1e1 was preferentially affected by ROR{alpha} expression, while Sult2a1 was influenced by both ROR{alpha} and ROR{gamma}. It also showed that RORs are negative modulators of Sult1e1 and Sult2a1 expression. The latter was supported by observations showing that transfection of WT ROR{alpha} or activated VP-ROR{alpha} suppressed the expression of endogenous Sult1e1 and Sult2a1 expression in mouse hepatocytes compared with cells transfected with empty vector (Fig. 5B).


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 5. Altered expression of Sult1e1 and Sult2a1 in DKO, ROR{alpha}sg/sg, and ROR{gamma}–/– mice. A: livers were collected at CT19 from 4 individual, 8- to 12-wk-old, male or female mice within each genotype group and then analyzed by QRT-PCR. B: suppression of Sult1e1 and Sul2a1 expression by exogeneous ROR{alpha} in normal hepatocytes. Mouse primary hepatocytes were transiently transfected with empty vector or expression vectors for ROR{alpha} or VP-ROR{alpha} (black bars). Total RNA was prepared 40 h after transfection. In a 2nd experiment, livers of female CD-1 mice were transfected with empty vector or expression vectors for ROR{alpha} or VP-ROR{alpha} by a hydrodynamic gene deliver method (gray bars). Six hours after transfection, mice were killed and total liver RNA was isolated. RNA was subjected to QRT-PCR analyses to detect the expression of endogenous Sult1e1 and Sult2a1 mRNA. The percent inhibition by ROR{alpha} was calculated and plotted. C: comparison of Sult1e1 and Sult2a1 activity in WT, ROR{alpha}sg/sg, and ROR{gamma}–/–, and DKO mice. Livers were collected at CT19 from 4 individual, 8- to 12-wk-old, male or female mice within each genotype group. Liver extracts were prepared and assayed for Sult1e1 and Sult2a1 activity as described in MATERIALS AND METHODS. *P < 0.05 (compared with WT).

 
To examine whether the increase in Sult1e1 and Sult2a1 expression was reflected in a corresponding increase in protein activity, we compared Sult activities in liver from WT, DKO, ROR{gamma}–/–, and ROR{alpha}sg/sg mice using, respectively, [3H]-labeled estradiol and dehydroepiandrosterone as substrates. As shown in Fig. 5C, Sult1e1 enzyme activity was greatly increased in liver extracts from DKO and ROR{alpha}sg/sg mice compared with those from WT and ROR{gamma}–/– mice. Sult1e1 activity was increased in liver of both male and female mice. In contrast, Sult2a1 activity was enhanced in liver extracts from DKO and ROR{gamma}–/– mice and to a lesser extent in ROR{alpha}sg/sg mice. These results show that the induction in Sult1e1 and Sult2a1 enzyme activities reflect the increased expression of their corresponding mRNAs as observed by QRT-PCR and Northern blot analysis.

To determine whether the changes in the expression of steroid metabolizing enzymes in liver of ROR-deficient mice had any effect on blood steroid levels, we analyzed estrogen and DHEA in blood from WT and ROR-deficient mice. This analysis indicated no significant differences in blood levels of estrogen and DHEA between male and female WT, ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice (data not shown).

Circadian pattern of expression of ROR-regulated genes.
Since ROR{alpha}1 and ROR{gamma}1 exhibit an oscillatory pattern of expression (Fig. 1B), one would expect that at least some genes regulated by these receptors show a similar or inverse oscillatory pattern of expression during circadian rhythm. We, therefore, examined the level of expression of Elovl3, Hsd3b5, and Sult1e1 in livers from WT male mice as a function of CT. As shown in Fig. 6, the expression of Elovl3, which is positively regulated by RORs, exhibited a rhythmicity that was in phase with that of RORs, while the expression of Sult1e1, which is suppressed by RORs, was out of phase with ROR expression. Hsd3b5 did not exhibit a clear rhythmic pattern of expression.


Figure 6
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 6. Circadian oscillation of the expression of Sult1e1, Hsd3b5, and Elovl3 mRNA in livers of WT male AKR/J mice. The mRNA expression was examined by QRT-PCR analysis and normalized relative to cyclophilin B. Data from a single 24 h period were double plotted. The mice used in the study were maintained on a constant 12-h light/12-h dark cycle as indicated by the bar.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main objective of this study was to obtain insights into the physiological roles of ROR{alpha} and ROR{gamma} in liver. Liver expresses the isoforms ROR{alpha}1, ROR{alpha}4, and ROR{gamma}1 (5, 14) of which ROR{alpha}1 and ROR{gamma}1 exhibit an oscillatory pattern of expression consistent with a circadian rhythm (Fig. 1). ROR{alpha}1 and ROR{gamma}1 are maximally expressed between CT16 and CT0. To study their function and investigate the possibility of functional redundancy between these two receptors, we generated ROR{alpha}sg/sgROR{gamma}–/– DKO mice. The DKO mice exhibited many of the same phenotypic changes reported for the single knockout mice. The DKO mice exhibited reduced triglyceride and cholesterol levels as previously reported for ROR{alpha}sg/sg mice (32). Triglyceride levels were not significantly changed in ROR{gamma}–/– mice; however, these mice exhibited reduced blood glucose levels. The latter was also observed in DKO mice. These observations suggest that RORs have distinct regulatory roles in triglyceride and glucose homeostasis.

To obtain further insight into the physiological functions of ROR{alpha} and ROR{gamma} in liver, we examined the gene expression profiles of livers from WT, ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice by microarray analysis (Table 2). Comparison of the gene expression profiles indicated that RORs affect the expression of a number of genes. This led to several important conclusions.

Firstly, the gene expression profiles showed that some genes were downregulated in livers from DKO mice compared with WT mice, while other genes were induced in DKO mice. These data suggest that ROR{alpha} and ROR{gamma} can influence gene expression in a positive as well as negative manner. This concept is in agreement with conclusions from recent crystal structure studies indicating that ROR receptors are ligand-dependent transcription factors (20, 38). In addition, RORs have been reported to interact with co-repressors, such as NCoR and RIP140, as well as co-activators, including CBP and SRC1 (12, 17). Although it is likely that RORs control the expression of some of these genes by an indirect mechanism, the positive and negative regulation of the transcription of at least some genes may involve an interaction of RORs with these co-repressors and co-activators. As has been demonstrated for the regulation of gene expression by ROR{alpha} in Purkinje cells, the promoter context of the RORE is important in determining what transcriptional mediators are recruited by RORs (12). This may also determine whether ROR induces or represses the expression of a gene.

In addition, our gene profile analyses demonstrated that the expression of certain genes is affected preferentially by ROR{alpha} or ROR{gamma}, while the expression of other genes is influenced by both receptors. For example, Sult1e1 and Cyp7b1 mRNA expression is affected preferentially in liver of ROR{alpha}sg/sg mice, while Elovl3 is suppressed preferentially in ROR{gamma}–/– mice. In contrast, Hsd3b5 is only slightly downregulated in livers of ROR{alpha}sg/sg and ROR{gamma}–/– mice, while it is dramatically repressed in DKO mice. Similarly, repression of Cyp8b1 expression was only observed in livers of DKO mice. These observations suggest that there is a degree of functional redundancy between ROR{alpha} and ROR{gamma}. The latter may not be surprising since previous studies have shown that although ROR{alpha} and ROR{gamma} receptors have distinct affinities for specific ROREs, they also can bind and compete for the same RORE (11, 29). Thus the regulation of expression of specific genes by ROR{alpha} and ROR{gamma} depends on their affinity to the respective RORE present in the target gene. In addition, the promoter context of the RORE likely plays an important role as well in determining which ROR receptor binds which RORE (4, 9, 12, 13, 17).

Most importantly, comparison of the gene expression profiles allowed us to identify categories of genes affected by the loss of ROR expression. This analysis suggested novel roles for RORs in the modulation of the expression of a number of phase I and II enzymes in the liver (Fig. 7). The liver is the primary site of drug metabolism that has been classified as phase I and phase II reactions. Phase I reactions are primarily mediated by P450 microsomal enzymes catalyzing oxidation and hydroxylation and several other enzymes flavin monooxygenases, peroxidases, dehydrogenases, oxidases, etc. (47). Sulfation, glucuronidation, and glutathione conjugation are the major classes of phase II metabolism. Often compounds undergo phase I oxidation before undergoing phase II conjugation. Phase I and II enzymes play an important role in the detoxification and elimination of endogenous, e.g., steroids, bile acids as well as exogenous compounds, e.g., xenobiotics, drugs, and environmental chemicals. However, Cyps also play a role in the generation of active metabolites.


Figure 7
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 7. Schematic view of the metabolic pathways affected in ROR{alpha}sg/sg, ROR{gamma}–/–, and DKO mice. ROR{alpha} and ROR{gamma} influence several phase I and phase II metabolic pathways, including bile acid biosynthesis, steroid, xenobiotic, and fatty acid metabolism. GST, glutathione-S-transferase; GSH, glutathione.

 
Several of the phase I genes affected by RORs play a role in bile acid biosynthesis (Fig. 7). Cholesterol homeostasis is a balance between the dietary intake of cholesterol, the endogenous biosynthesis of cholesterol, and the disposal of cholesterol in the form of bile acids. The classic or neutral pathway of bile acid biosynthesis involves the conversion of cholesterol to 7{alpha}-hydroxycholesterol by the rate-limiting enzyme Cyp7a1 and its further conversion by several other enzymes, including Cyp27a1 and Cyp8b1, into cholic acid (CA) and chenodeoxycholic acid. The alternative (acidic) bile acid synthesis pathway involves Cyp27a1 and Cyp7b1 that mediate the 7{alpha}-hydroxylation of the oxysterols 25- and 27-hydroxycholesterol and several steroids. Our gene profiling analysis showed that RORs affect Cyp7b1 and Cyp8b1 expression, suggesting a role for RORs in the control of both the neutral and the acidic pathway of bile acid biosynthesis. RORs did not significantly affect the expression of Cyp7a1 and Cyp27a1 (data not shown), which have major functions in bile acid biosynthesis. In addition to P450 enzymes involved in bile acid biosynthesis, RORs also influence the expression of a number of other phase I enzymes, including members of the Cyp4a family, which catalyze {omega}-hydroxylation of fatty acids and related compounds facilitating their degradation (40), and members of the Cyp2b family, which are involved in the oxidation of xenobiotics (44). These results further support the conclusion that RORs affect the expression of Cyp7b1 and Cyp2b10 and play a role in the modulation of phase I metabolic pathways.

RORs also regulate the expression of a number of phase II metabolic enzymes, including several glutathione transferases and Sults (47). Glutathione transferases catalyze the conjugation of glutathione with a wide variety of xenobiotics generally resulting in their detoxification and elimination (28). Sult1e1 is main enzyme for sulfonation of estradiol, estrone, and genistein. Sulfonation generally increases the aqueous solubility of substrates and causes a loss of biological activity because sulfonated steroids are unable to bind their (nuclear) receptors. Sult1e1 appears to be regulated preferentially by ROR{alpha} (Fig. 7). Sult2a1 catalyzes the sulfonation of procarcinogens, xenobiotics, hydroxysteroids, and bile acids (23). Sult2a1 is important for the sulfonation of DHEA and LCA. Sult2a1 expression is affected by both ROR{alpha} and ROR{gamma}. Exogenous expression of ROR{alpha} in mouse hepatocytes suppressed the expression of Sult1e1 and Sult2a1 in agreement with the concept that RORs are negative modulators of the Sult1e1 expression. Another group of ROR-regulated genes involved in steroid metabolism are members of the Hsd3b and Hsd17b family (35). The expression of Hsd3b4 and Hsd3b5, which are involved in the inactivation of steroid hormones, is greatly diminished in DKO mice and appears to be regulated by both ROR{alpha} and ROR{gamma}. These observations strongly suggest that RORs modulate several aspects of steroid and bile acid metabolism. In this respect it is interesting to note that cholesterol sulfate has been reported to bind the ROR{alpha} receptor (19). Possibly other sulfated steroid or cholesterol metabolites may function as ligands for RORs. Such an interaction could lead to repression of genes (e.g., Sults) regulated by RORs thereby creating a feedback control mechanism. To test this idea further, we analyzed the binding of estrogen- and estrone sulfate, products of Sult1e1, to ROR{alpha}; however, no binding of these compounds was detectable (Jetten AM, unpublished observations).

ROR{alpha}1 and ROR{gamma}1, but not ROR{alpha}4, exhibit an oscillatory pattern of expression in liver consistent with a circadian rhythm. Therefore, one might expect that the expression of genes positively regulated by ROR{alpha}1 and ROR{gamma}1 is in phase with ROR expression and that the inverse is true for genes suppressed by RORs. This is supported by observations showing that the expression of Elovl3, which is positively regulated by ROR{gamma}1, is in phase with the circadian pattern of ROR{gamma}1 and that the expression of Sult1e1, which is negatively affected by ROR{alpha}, is out of phase with the circadian pattern of expression of ROR{alpha}1 in agreement with the concept that it is under the control of ROR{alpha}1. The expression of Hsd3b5 did not exhibit a clear circadian pattern, and its regulation might involve a different mechanism possibly involving ROR{alpha}4. These results are in agreement with previous observations showing a role for RORs in the regulation of gene expression during circadian rhythm (17). It is interesting to note that the phase of the circadian profile of RORs in liver is the inverse of that reported for the Rev-erb nuclear receptors, which function as negative regulators of transcription (49). Consequently, Rev-erb receptors might be involved in the negative regulation of ROR{alpha}1 and ROR{gamma}1 expression. In addition, Rev-erb receptors have been reported to compete with RORs for binding to ROREs (3). Thus, during circadian rhythm ROR and Rev-erb receptors might function as positive and negative regulators of overlapping sets of genes.

Likely not all genes identified in Table 2 are regulated by RORs by a direct mechanism; some may be controlled by an indirect mechanism possibly involving regulation by other transcription factors that themselves are controlled by RORs. Previous studies have identified several genes, including ApoA5, that are regulated directly by ROR{alpha} (26, 27). In this study we show that Cyp7b1 expression is suppressed in DKO and ROR{alpha}sg/sg mice. The Cyp7b1 gene contains an RORE at nt –952 in the upstream promoter region that was able to bind ROR{alpha} and mediate transcriptional activation by ROR{alpha} (Wada T, Kang HS, Angers M, Gong H, Bhatia S, Khadem S, Ren S, Ellis E, Strom SC, Jetten AM, Xie W, unpublished observations). In addition, chromatin immunoprecipitation analysis showed that this RORE binds ROR{alpha} in vivo suggesting that Cyp7b1 is a direct target gene of ROR{alpha}. Interestingly, many of the phase I and II genes found to be affected by RORs have been previously reported to be targets for transcriptional regulation by other nuclear receptors, including the liver X receptor (LXR), constitutive androstane receptor (CAR or NR1I3), pregnane X receptor (PXR), hepatocyte nuclear factor 4{alpha} (HNF4{alpha}), peroxisome proliferator-activated receptors, and farnesoid X receptor (FXR) (1, 6, 18, 24, 31, 33). For example, Sult2a1 expression is under the transcriptional control of CAR, PXR, and FXR, while CAR and PXR function, respectively, as a negative and positive regulator of Cyp2b10 expression, and HNF4{alpha} is a transcriptional activator of several Cyps, including Cyp8b1. The effects of RORs on gene expression could involve regulation of the expression of one or more of these receptors, synthesis of receptor ligands or competition for the same DNA response element. Examination of CAR and LXR{alpha} expression showed no significance difference in their expression between WT and ROR-deficient mice (Kang HS, unpublished observations). Future studies have to determine whether there is any cross talk between these receptors and RORs as has been recently demonstrated for CAR and HNF4{alpha} (8).

In summary, our study demonstrates that ROR{alpha}1 and ROR{gamma} exhibit an oscillatory pattern of expression consistent with a circadian profile. Analysis of the gene expression profiles indicated that ROR{alpha} and ROR{gamma} affect downstream gene expression in a positive and negative manner. Some genes were modulated preferentially by ROR{alpha} or by ROR{gamma}, while a number of genes were affected by both ROR{alpha} and ROR{gamma}, suggesting functional redundancy. Most importantly, our study demonstrates that RORs are able to modulate the expression a number of phase I and phase II metabolic enzymes, suggesting that expression of RORs can affect bile biosynthesis and the metabolism of steroids and xenobiotics.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the Intramural Research Program of the NIEHS, NIH, and by Clinical Nutrition Research Unit Center Grant 1P30 DK-072476 entitled "Nutritional Programming: Environmental and Molecular Interactions" sponsored by National Institute of Diabetes and Digestive and Kidney Diseases (J. M. Gimble).


   ACKNOWLEDGMENTS
 
The authors thank Dr. C. Teng for valuable comments on the manuscript. We thank Laura Miller for assistance with the mice.


   FOOTNOTES
 
Address for reprint requests and other correspondence: A. M. Jetten, Cell Biology Section, Div. of Intramural Research, The National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (e-mail: jetten{at}niehs.nih.gov).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

* H. S. Kang and M. Angers contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akiyama TE, Gonzalez FJ. Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors. Biochim Biophys Acta 1619: 223–234, 2003.[Medline]
  2. Andre E, Conquet F, Steinmayr M, Stratton SC, Porciatti V, Becker-Andre M. Disruption of retinoid-related orphan receptor beta changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice. EMBO J 17: 3867–3877, 1998.[CrossRef][Web of Science][Medline]
  3. Austin S, Medvedev A, Yan ZH, Adachi H, Hirose T, Jetten AM. Induction of the nuclear orphan receptor RORgamma during adipocyte differentiation of D1 and 3T3–L1 cells. Cell Growth Differ 9: 267–276, 1998.[Abstract]
  4. Boukhtouche F, Doulazmi M, Frederic F, Dusart I, Brugg B, Mariani J. RORalpha, a pivotal nuclear receptor for Purkinje neuron survival and differentiation: from development to ageing. Cerebellum 5: 97–104, 2006.[CrossRef][Web of Science][Medline]
  5. Chauvet C, Bois-Joyeux B, Danan JL. Retinoic acid receptor-related orphan receptor (ROR) alpha4 is the predominant isoform of the nuclear receptor RORalpha in the liver and is up-regulated by hypoxia in HepG2 human hepatoma cells. Biochem J 364: 449–456, 2002.[CrossRef][Web of Science][Medline]
  6. Cheung C, Akiyama TE, Kudo G, Gonzalez FJ. Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNF1alpha)-deficient mice. Biochem Pharmacol 66: 2011–2020, 2003.[CrossRef][Web of Science][Medline]
  7. Delerive P, Monte D, Dubois G, Trottein F, Fruchart-Najib J, Mariani J, Fruchart JC, Staels B. The orphan nuclear receptor ROR alpha is a negative regulator of the inflammatory response. EMBO Rep 2: 42–48, 2001.[CrossRef][Web of Science][Medline]
  8. Ding X, Lichti K, Kim I, Gonzalez FJ, Staudinger JL. Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha. J Biol Chem 281: 26540–26551, 2006.[Abstract/Free Full Text]
  9. Dussault I, Fawcett D, Matthyssen A, Bader JA, Giguere V. Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer. Mech Dev 70: 147–153, 1998.[CrossRef][Web of Science][Medline]
  10. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgammat in the generation of fetal lymphoid tissue inducer cells. Nat Immun 5: 64–73, 2004.[CrossRef][Web of Science]
  11. Giguere V, Beatty B, Squire J, Copeland NG, Jenkins NA. The orphan nuclear receptor ROR alpha (RORA) maps to a conserved region of homology on human chromosome 15q21-q22 and mouse chromosome 9. Genomics 28: 596–598, 1995.[CrossRef][Web of Science][Medline]
  12. Gold DA, Baek SH, Schork NJ, Rose DW, Larsen DD, Sachs BD, Rosenfeld MG, Hamilton BA. RORalpha coordinates reciprocal signaling in cerebellar development through sonic hedgehog and calcium-dependent pathways. Neuron 40: 1119–1131, 2003.[CrossRef][Web of Science][Medline]
  13. Hamilton BA, Frankel WN, Kerrebrock AW, Hawkins TL, FitzHugh W, Kusumi K, Russell LB, Mueller KL, van Berkel V, Birren BW, Kruglyak L, Lander ES. Disruption of the nuclear hormone receptor RORalpha in staggerer mice. Nature 379: 736–739, 1996.[CrossRef][Medline]
  14. Hirose T, Smith RJ, Jetten AM. ROR gamma: the third member of ROR/RZR orphan receptor subfamily that is highly expressed in skeletal muscle. Biochem Biophys Res Commun 205: 1976–1983, 1994.[CrossRef][Web of Science][Medline]
  15. Jaradat M, Stapleton C, Tilley SL, Dixon D, Erikson CJ, McCaskill JG, Kang HS, Angers M, Liao G, Collins J, Grissom S, Jetten AM. Modulatory role for retinoid-related orphan receptor alpha in allergen-induced lung inflammation. Am J Respir Crit Care Med 174: 1299–309, 2006.[Abstract/Free Full Text]
  16. Jetten AM. Recent advances in the mechanisms of action and physiological functions of the retinoid-related orphan receptors (RORs). Curr Drug Targets Inflamm Allergy 3: 395–412, 2004.[CrossRef][Medline]
  17. Jetten AM, Joo JH. Retinoid-related orphan receptors (RORs): roles in cellular differentiation and development. Adv Devel Biol 16: 314–354, 2006.
  18. Kalaany NY, Mangelsdorf DJ. LXRS and FXR: the yin and yang of cholesterol and fat metabolism. Annu Rev Physiol 68: 159–191, 2006.[CrossRef][Web of Science][Medline]
  19. Kallen J, Schlaeppi JM, Bitsch F, Delhon I, Fournier B. Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A. J Biol Chem 279: 14033–14038, 2004.[Abstract/Free Full Text]
  20. Kallen JA, Schlaeppi J, Bitsch F, Geisse S, Geiser M, Delhon I, Fournier B. X-ray structure of the RORa LBD at 1.63A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORa. Structure 10: 1697–1707, 2002.[Medline]
  21. Kang HS, Beak JY, Kim YS, Petrovich RM, Collins JB, Grissom SF, Jetten AM. NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein. Biochem J 397: 89–99, 2006.[CrossRef][Web of Science][Medline]
  22. Kim MS, Shigenaga J, Moser A, Grunfeld C, Feingold KR. Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response. Am J Physiol Endocrinol Metab 287: E731–E738, 2004.[Abstract/Free Full Text]
  23. Kitada H, Miyata M, Nakamura T, Tozawa A, Honma W, Shimada M, Nagata K, Sinal CJ, Guo GL, Gonzalez FJ, Yamazoe Y. Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity. J Biol Chem 278: 17838–17844, 2003.[Abstract/Free Full Text]
  24. Kliewer SA. The nuclear pregnane X receptor regulates xenobiotic detoxification. J Nutr 133: 2444S–2447S, 2003.[Abstract/Free Full Text]
  25. Kurebayashi S, Ueda E, Sakaue M, Patel DD, Medvedev A, Zhang F, Jetten AM. Retinoid-related orphan receptor gamma (RORgamma) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc Natl Acad Sci USA 97: 10132–10137, 2000.[Abstract/Free Full Text]
  26. Lau P, Nixon SJ, Parton RG, Muscat GE. RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR. J Biol Chem 279: 36828–36840, 2004.[Abstract/Free Full Text]
  27. Lind U, Nilsson T, McPheat J, Stromstedt PE, Bamberg K, Balendran C, Kang D. Identification of the human ApoAV gene as a novel RORalpha target gene. Biochem Biophys Res Commun 330: 233–241, 2005.[CrossRef][Web of Science][Medline]
  28. McCarver DG, Hines RN. The ontogeny of human drug-metabolizing enzymes: phase II conjugation enzymes and regulatory mechanisms. J Pharmacol Exp Ther 300: 361–366, 2002.[Abstract/Free Full Text]
  29. Medvedev A, Yan ZH, Hirose T, Giguere V, Jetten AM. Cloning of a cDNA encoding the murine orphan receptor RZR/ROR gamma and characterization of its response element. Gene 181: 199–206, 1996.[CrossRef][Web of Science][Medline]
  30. Meyer T, Kneissel M, Mariani J, Fournier B. In vitro and in vivo evidence for orphan nuclear receptor RORalpha function in bone metabolism. Proc Natl Acad Sci USA 97: 9197–9202, 2000.[Abstract/Free Full Text]
  31. Qatanani M, Moore DD. CAR, the continuously advancing receptor, in drug metabolism and disease. Curr Drug Metab 6: 329–339, 2005.[CrossRef][Web of Science][Medline]
  32. Raspe E, Duez H, Gervois P, Fievet C, Fruchart JC, Besnard S, Mariani J, Tedgui A, Staels B. Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha. J Biol Chem 276: 2865–2871, 2001.[Abstract/Free Full Text]
  33. Runge-Morris M, Kocarek TA. Regulation of sulfotransferases by xenobiotic receptors. Curr Drug Metab 6: 299–307, 2005.[CrossRef][Web of Science][Medline]
  34. Schaeren-Wiemers N, Andre E, Kapfhammer JP, Becker-Andre M. The expression pattern of the orphan nuclear receptor RORbeta in the developing and adult rat nervous system suggests a role in the processing of sensory information and in circadian rhythm. Eur J Neurosci 9: 2687–2701, 1997.[CrossRef][Web of Science][Medline]
  35. Simard J, Ricketts ML, Gingras S, Soucy P, Feltus FA, Melner MH. Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family. Endocr Rev 26: 525–582, 2005.[Abstract/Free Full Text]
  36. Srinivas M, Ng L, Liu H, Jia L, Forrest D. Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor beta. Mol Endocrinol 20: 1728–1741, 2006.[Abstract/Free Full Text]
  37. Stapleton CM, Jaradat M, Dixon D, Kang HS, Kim SC, Liao G, Carey MA, Cristiano J, Moorman MP, Jetten AM. Enhanced susceptibility of staggerer (RORalphasg/sg) mice to lipopolysaccharide-induced lung inflammation. Am J Physiol Lung Cell Mol Physiol 289: L144–L152, 2005.[Abstract/Free Full Text]
  38. Stehlin C, Wurtz JM, Steinmetz A, Greiner E, Schule R, Moras D, Renaud JP. X-ray structure of the orphan nuclear receptor RORbeta ligand-binding domain in the active conformation. EMBO J 20: 5822–5831, 2001.[CrossRef][Web of Science][Medline]
  39. Stehlin-Gaon C, Willmann D, Zeyer D, Sanglier S, Van Dorsselaer A, Renaud JP, Moras D, Schule R. All-trans retinoic acid is a ligand for the orphan nuclear receptor RORbeta. Nat Struct Biol 10: 820–825, 2003.[CrossRef][Web of Science][Medline]
  40. Sue Masters B, Marohnic CC. Cytochromes P450–a family of proteins and scientists-understanding their relationships. Drug Metab Rev 38: 209–225, 2006.[CrossRef][Medline]
  41. Sun Z, Unutmaz D, Zou YR, Sunshine MJ, Pierani A, Brenner-Morton S, Mebius RE, Littman DR. Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288: 2369–2373, 2000.[Abstract/Free Full Text]
  42. Ueda E, Kurebayashi S, Sakaue M, Backlund M, Koller B, Jetten AM. High incidence of T-cell lymphomas in mice deficient in the retinoid-related orphan receptor RORgamma. Cancer Res 62: 901–909, 2002.[Abstract/Free Full Text]
  43. Vu-Dac N, Gervois P, Grotzinger T, De Vos P, Schoonjans K, Fruchart JC, Auwerx J, Mariani J, Tedgui A, Staels B. Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORalpha. J Biol Chem 272: 22401–22404, 1997.[Abstract/Free Full Text]
  44. Wang H, Negishi M. Transcriptional regulation of cytochrome p450 2B genes by nuclear receptors. Curr Drug Metab 4: 515–525, 2003.[CrossRef][Web of Science][Medline]
  45. Weng L, Dai H, Zhan Y, He Y, Stepaniants SB, Bassett DE. Rosetta error model for gene expression analysis. Bioinformatics 22: 1111–11121, 2006.[Abstract/Free Full Text]
  46. Yang X, Downes M, Yu RT, Bookout AL, He W, Straume M, Mangelsdorf DJ, Evans RM. Nuclear receptor expression links the circadian clock to metabolism. Cell 126: 801–810, 2006.[CrossRef][Web of Science][Medline]
  47. Zamek-Gliszczynski MJ, Hoffmaster KA, Nezasa K, Tallman MN, Brouwer KL. Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites. Eur J Pharm Sci 27: 447–486, 2006.[CrossRef][Web of Science][Medline]
  48. Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, Ren S, Evans RM, Xie W. A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J Biol Chem 281: 15013–15020, 2006.[Abstract/Free Full Text]
  49. Zvonic S, Ptitsyn AA, Conrad SA, Scott LK, Floyd ZE, Kilroy G, Wu X, Goh BC, Mynatt RL, Gimble JM. Characterization of peripheral circadian clocks in adipose tissues. Diabetes 55: 962–970, 2006.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
H. S. Kang, J. Y. Beak, Y.-S. Kim, R. Herbert, and A. M. Jetten
Glis3 Is Associated with Primary Cilia and Wwtr1/TAZ and Implicated in Polycystic Kidney Disease
Mol. Cell. Biol., May 15, 2009; 29(10): 2556 - 2569.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
Y. Chen, S. Coulter, A. M. Jetten, and J. A. Goldstein
Identification of Human CYP2C8 as a Retinoid-Related Orphan Nuclear Receptor Target Gene
J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 192 - 201.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Y. Tomaru, M. Nakanishi, H. Miura, Y. Kimura, H. Ohkawa, Y. Ohta, Y. Hayashizaki, and M. Suzuki
Identification of an inter-transcription factor regulatory network in human hepatoma cells by Matrix RNAi
Nucleic Acids Res., March 1, 2009; 37(4): 1049 - 1060.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
V. Mongrain, X. Ruan, H. Dardente, E. E. Fortier, and N. Cermakian
Clock-dependent and independent transcriptional control of the two isoforms from the mouse Rorgammagene.
Genes Cells, December 1, 2008; 13(12): 1197 - 1210.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
T. Wada, H. S. Kang, A. M. Jetten, and W. Xie
The Emerging Role of Nuclear Receptor ROR{alpha} and Its Crosstalk with LXR in Xeno- and Endobiotic Gene Regulation
Experimental Biology and Medicine, October 1, 2008; 233(10): 1191 - 1201.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
T. Wada, H. S. Kang, M. Angers, H. Gong, S. Bhatia, S. Khadem, S. Ren, E. Ellis, S. C. Strom, A. M. Jetten, et al.
Identification of Oxysterol 7{alpha}-Hydroxylase (Cyp7b1) as a Novel Retinoid-Related Orphan Receptor {alpha} (ROR{alpha}) (NR1F1) Target Gene and a Functional Cross-Talk between ROR{alpha} and Liver X Receptor (NR1H3)
Mol. Pharmacol., March 1, 2008; 73(3): 891 - 899.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
31/2/281    most recent
00098.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kang, H. S.
Right arrow Articles by Jetten, A. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kang, H. S.
Right arrow Articles by Jetten, A. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.